Interpretive Summary: Cryptosporidium parvum oocysts were recovered from 10L volumes of water, both source and finished, using continuous flow centrifugation and immunomagnetic separation. To determine recovery, bioassay and nested PCR were used for viability. Fluorescence in situ hybridization probes were used to detect the oocysts. The best oocyst recoveries occurred when using source water. These methods appear to be both more rapid and economical when compared to traditional methods of oocyst recovery from water samples.

Technical Abstract:
Continuous flow centrifugation (CFC) was used in conjunction with immunomagnetic separation (IMS) to recover oocysts of Cryptosporidium parvum from 10 L volumes of source and finished water samples. Using a spiking dose of 1 x 105 oocysts, 25% - 106% of spiked oocysts were recovered, with the best recoveries from source water samples. Recovered oocysts were viable, as determined by bioassay using HCT-8 cells and nested PCR, and were detectable using fluorescence in situ hybridization (FISH) probes directed against the 18S rRNA gene. Using a spiking dose of 1 x 102 oocysts in 10 L of source water, oocysts were recovered in 9 of 10 trials as determined by IFA microscopy, with a mean oocyst recovery of 4.4%. Another 9 of 10 trials were positive by nested PCR for the C. parvum Cp41 and TRAP C-1 genes. No oocysts were detected in unspiked controls using IFA microscopy or nested PCR. CFC and IMS may provide a more rapid and economical alternative to traditional, capsule filtration-based methods for isolation of C. parvum oocysts from water samples.